Construction of TNF-binding proteins by grafting hypervariable regions of F10 antibody on human fibronectin domain scaffold

Abstract

Tumor necrosis factor (TNF) plays a key role in the pathogenesis of various diseases. To study the possibility of constructing TNF-binding proteins by grafting hypervariable regions of immunoglobulins (CDR), we have replaced amino acid sequences of loops from the tenth type III domain of human fibronectin ((10)Fn3) by amino acid sequences of CDR from the light and heavy chains of the anti-TNF antibody F10. The assessment of TNF-binding properties of the resulting proteins by ELISA has revealed the highest activity of Hd3 containing sequences CDR-H1 and CDR-H2 of the antibody F10 and of Hd2 containing sequences CDR-H1 and CDR-H3. The proteins constructed by us on the fibronectin domain scaffold specifically bound TNF during Western blotting and also weakened its cytotoxic effect on L929 line cells. The highest neutralizing activity was demonstrated by the proteins Hd2 and Hd3, which induced, respectively, 10- and 50-fold increase in the EC(50) of TNF.

Functional Consequences of Complementarity-determining Region Deactivation in a Multifunctional Anti-nucleic Acid Antibody

“…A full understanding of the role of the 3D8 LCDR1 domain in mAb function could be obtained by investigating whether the transfer of this domain into a wide range of mAbs results in their functionalization. In the protein engineering field, functional CDRs derived from Abs have been grafted into non-Ab scaffold proteins such as GFP, neocarzinostatin, and human fibronectin to create functionalized proteins (33–35). It may be worthwhile grafting the 3D8 LCDR1 domain onto these non-Ab scaffolds.…”

The Effect of TNF and VEGF on the Properties of Ea.hy926 Endothelial Cells in a Model of Multi-Cellular Spheroids

“…The tumor necrosis factor alpha (TNF-α) has been shown to play the key role in the development and maintenance of inflammation in diseases, such as rheumatoid arthritis, psoriasis, Crohn’s disease, etc. [ 1 , 2 ]. Both the inflammatory process and tumor growth are accompanied by tissue hypoxia, which leads to the formation of new blood vessels under the influence of the vascular endothelial growth factor (VEGF), which is secreted by epithelial cells in conditions of hypoxia [ 3 , 4 ].…”